Generalized stochastic super-resolution sequencing

ABSTRACT

A method of sequencing a plurality of polynucleotides includes: attaching a single DNA template molecule to each of a plurality of attachment elements on a sample container, wherein the average distance between adjacent elements is less than Abbe&#39;s limit; applying a stochastic photo-switching chemistry to all of the molecules at the same time to cause the attached molecules to fluoresce in on and off events in up to four different colors by stochastic photo-switching; and imaging the on and off events in a color channel for each color in real-time as the on and off events are occurring for the attached molecules.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/640,909, entitled “GENERALIZED STOCHASTIC SUPER-RESOLUTIONSEQUENCING”, and filed on Mar. 9, 2018. The specification of which isdisclosed herein by reference.

BACKGROUND

Numerous technologies in the field of biology, including those used inDNA sequencing, have benefited from improved imaging systems andtechniques. Early approaches to DNA sequencing included the dideoxychain termination method (i.e., Sanger sequencing) and the chemicaldegradation method (i.e., Maxam-Gilbert sequencing). A desire for alower-cost and more rapid alternative to these techniques led to thedevelopment of an ensemble sequencing approach known as Sequencing bySynthesis (SBS). In this process, single template molecules are firstchemically amplified to generate surface-bound “clusters” of moleculeshaving the same sequence. Once the clusters are produced, sequencingbegins whereby fluorescent nucleotides are added by a modifiedpolymerase based on the sequence of the template. The clusters are thenexcited by a light source resulting in the emittance of a characteristicfluorescent signal to determine the base call. The dyes are thenremoved, along with a 3′ chain terminator, and the cycle is repeated forthe next base in the sequence.

SUMMARY

Various examples of the technologies disclosed herein provide methodsand techniques for super-resolution sequencing. In one example, a systemand method for sequencing a plurality of polynucleotides includes:attaching a single DNA template molecule to each of a plurality ofattachment elements on a sample container, wherein the average distancebetween adjacent elements is less than Abbe's limit; applying astochastic photo-switching chemistry to all of the molecules at the sametime to cause the attached molecules to fluoresce in on and off eventsin up to four different colors by stochastic photo-switching; andimaging the on and off events in a color channel for each color inreal-time as the on and off events are occurring for the attachedmolecules. The average distance between adjacent elements may be lessthan about 20 nm or it may be within a range of about 2 nm to about 20nm.

In a further example, a method of sequencing polynucleotides mayinclude: providing an array of nucleotide sequences anchored to a solidsupport, wherein the average distance between adjacent anchors is lessthan Abbe's limit; providing a mixture to the array comprising an enzymecapable of coupling nucleotides, a deblocking agent, a nucleic acidbound to a strand of nucleotides having a sequence complimentary to thenucleotide sequences anchored to the solid support, and more than onenucleotide analog comprising a base with a label moiety andcorresponding quencher moiety bound thereto wherein the label moietiesare correlated with a specific base moiety; and allowing sequentialaddition of a plurality of the nucleotide analogs to the nucleic acid toproceed via several reaction cycles in the mixture while concurrentlyimaging the label moieties within the array; wherein each reaction cyclemay include: (i) the polymerase adding a nucleotide analog to thenucleic acid by cleaving the quencher moiety and forming a transientnucleic acid species comprising the label moiety; and (ii) thedeblocking agent modifying the transient nucleic acid species to removethe label moiety. In some applications, the average distance betweenadjacent anchors is less than 20 nm. In further applications, theaverage distance between adjacent anchors is within a range of 2 nm to20 nm. The enzyme capable of coupling nucleotides may include apolymerase, a myosin or a kinase.

The nucleotide analog may include a pentose moiety having a 3′ carbonand the label moiety may be attached to the nucleotide at the 3′ carbon.In another example, the nucleotide analog may include a triphosphatemoiety and the quencher moiety may be attached to the triphosphatemoiety.

In some applications, the deblocking agent may include a phosphoesteraseenzyme (e.g., phosphodiesterase, phosphotriesterase, etc.). Thephosphoesterase may be included to selectively remove a phosphodiestermoiety or the phosphotriester moiety from the transient nucleic acidspecies. The phosphoesterase may be selected from the group consistingof Endonuclease IV and AP endonuclease.

As an example, the transient nucleic acid species may be present for atleast 1 millisecond before the deblocking agent modifies the transientnucleic acid species to remove the label moiety. As a further example,the transient nucleic acid species may be present for no more than 30seconds before the deblocking agent modifies the transient nucleic acidspecies to remove the label moiety.

In various applications, the several reaction cycles may include atleast 100 reaction cycles, whereby the nucleic acid is extended byaddition of at least 100 nucleotide analogs. In various applications,the enzyme capable of coupling nucleotides comprises a polymerase, amyosin, or a kinase.

In further examples, a method of sequencing a plurality ofpolynucleotides includes: attaching a single DNA template molecule toeach of a plurality of attachment elements on a sample container,wherein the average distance between adjacent elements is less thanAbbe's limit; applying a stochastic photo-switching chemistry to all ofthe molecules at the same time to cause the attached molecules tofluoresce in on and off events in up to four different colors bystochastic photo-switching; and imaging the on and off events in a colorchannel for each color in real-time as the on and off events areoccurring for the attached molecules. Due to the stochastic nature ofthe photo switching, in various examples the probability that an onevent for a given base for a given molecule will occur at the same timeas an on event for the same base at a molecule adjacent to the givenmolecule is less than 0.5%. The concentrations of reagents for thestochastic photo-switching may be chosen such that the probability thatan on event for a given base for a given molecule will occur at the sametime as an on event for the same base at a molecule adjacent to thegiven molecule is less than 0.5%. In other examples, otherconcentrations may be used. The average distance between adjacentelements may be less than about 20 nm or it may be within a range ofabout 2 nm to about 20 nm. In some applications, each of the pluralityof attachment elements on the sample container may be within a field ofview of an imager used to image the on and off events such that imagingof the on and off events occurs at the same time for the attachedmolecules at the plurality of attachment elements. Applying a stochasticphoto-switching chemistry to all of the attached molecules at the sametime may include applying a stochastic optical reconstructionmicroscopy, a DNA Points Accumulation for Imaging in NanoscaleTopography, or a direct stochastic optical reconstruction microscopystochastic photoswitching chemistry to all of the molecules at the sametime.

The process may further include controlling a rate at which the on andoff events occur to control a probability that an on event for a givenbase for a given molecule will occur at the same time as an on event forthe same base at a molecule adjacent to the given molecule. In someapplications, controlling the rate at which the on and off events occurmay include adjusting concentrations of nucleotides and enzyme in thestochastic photo-switching chemistry. In other applications, controllingthe rate at which the on and off events occur comprises adjusting the onand off times so that the probability that an on event for a given basefor a given molecule will occur at the same time as an on event for thesame base at a molecule adjacent to the given molecule is lower than adetermined error rate in a sequencing application in which the method isapplied.

In some applications, the process may further include determiningwhether an illumination intensity of a detected on event in a colorchannel is greater than a predetermined threshold. The process may alsoinclude determining whether a spot size of a detected on event in acolor channel is greater than a predetermined threshold.

An imaging system may include a sample container comprising a pluralityof attachment elements wherein a single DNA template molecule isattached to each of the attachment elements, and further wherein theaverage distance between adjacent attachment elements is less thanAbbe's limit; and an imager positioned to image photo-switchingoccurring at the plurality of attachment elements by capturing on andoff events in a plurality of color channels at the same time as the onand off events are occurring for the attached molecules when astochastic photo-switching chemistry is applied to all of the attachedmolecules at the same time causing the attached molecules to fluorescein the on and off events in up to four different colors. The samplecontainer may include a flowcell that comprises the plurality ofattachment elements at a plurality of sample locations. In variousexamples the average distance between adjacent elements is less thanabout 20 nm or it may be within a range of about 2 nm to about 20 nm. Invarious examples, each of the plurality of attachment elements on thesample container is within a field of view of the imager used to imagethe photo-switching such that the capturing of the on and off eventsoccurs at the same time for the attached molecules at the plurality ofattachment elements. The stochastic photo-switching chemistry applied toall of the attached molecules at the same time may include a stochasticoptical reconstruction microscopy, a DNA Points Accumulation for Imagingin Nanoscale Topography, or a direct stochastic optical reconstructionmicroscopy stochastic photoswitching chemistry.

The imaging system may employ stochastic optical reconstructionmicroscopy, DNA Points Accumulation for Imaging in Nanoscale Topographyor direct switching driven by photochemical reactions as the stochasticphoto-switching chemistry applied to cause the attached molecules tofluoresce. The concentrations of reagents for the stochastic photoswitching are such that the probability that an on event for a givenbase for a given molecule will occur at the same time as an on event forthe same base at a molecule adjacent to the given molecule is less than0.5%.

The imaging system may be implemented such that the rate at which the onand off events occur yields a probability that an on event for a givenbase for a given molecule will occur at the same time as an on event forthe same base at a molecule adjacent to the given molecule is lower thana determined error rate in a sequencing application in which the methodis applied. The imaging system may further determine whether anillumination intensity or a spot size of a detected on event in a colorchannel is greater than a predetermined threshold.

Other features and aspects of the disclosed technology will becomeapparent from the following detailed description, taken in conjunctionwith the accompanying drawings, which illustrate, by way of example, thefeatures in accordance with implementations of the disclosed technology.The summary is not intended to limit the scope of any inventionsdescribed herein, which are defined by the claims and equivalents.

It should be appreciated that all combinations of the foregoing concepts(provided such concepts are not mutually inconsistent) are contemplatedas being part of the inventive subject matter disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology disclosed herein, in accordance with one or moreexamples, is described in detail with reference to the followingfigures. These figures are provided to facilitate the reader'sunderstanding of the disclosed technology, and are not intended to beexhaustive or to limit the disclosure to the precise forms disclosed.Indeed, the drawings in the figures are provided for purposes ofillustration only, and merely depict typical or example examples of thedisclosed technology. Furthermore, it should be noted that for clarityand ease of illustration, the elements in the figures have notnecessarily been drawn to scale.

FIG. 1 illustrates a simplified block diagram of one example of an imagescanning system with which systems and methods disclosed herein may beimplemented.

FIG. 2 illustrates an example of a sample container including aplurality of attachment points, or anchors.

FIG. 3 illustrates an example of a side view of a row of the containerillustrated in FIG. 2 in the context of a particular stochasticphoto-switching chemistry.

FIG. 4 illustrates an example process for super-resolution sequencing.

FIG. 5 illustrates an example of ratchet biochemistry components thatcan be used in the super-resolution sequencing examples describedherein.

FIG. 6 illustrates an example of the chemical process upon theincorporation of the nucleotides

FIG. 7 illustrates an example of the process of super-resolution imagingusing the above-described chemical process.

It should be understood that the disclosed technology can be practicedwith modification and alteration, and that the disclosed technology belimited only by the claims and the equivalents thereof.

DETAILED DESCRIPTION

Various examples disclosed herein provide a super-resolution sequencingusing a homogenous, single-pot reaction in which a stochastic switchingof fluorophores is coupled to the sequencing reaction itself and imagingoccurs for multiple adjacent molecules in real time at the time of theincorporation. Particularly, in some applications, a sample container isprovided with a plurality of wells, or attachment elements, disposed ata spacing that is less than the spacing otherwise permitted to allowresolving the individual elements under Abbe's limit. A single DNAtemplate molecule is attached to each attachment element in the samplecontainer for sequencing. A sequencing chemistry is applied thatprovides a stochastic photo-switching of all the molecules in a group ofmolecules being imaged. Imaging of the reactions takes place inreal-time for all the molecules at the same time as the fluorophoresswitch on and off within the group. Because the reactions are stochasticand not synchronized among the molecules, there is a statisticalprobability that adjacent molecules will not incorporate the same baseat the same time.

In some applications, sequencing occurs by a polymerase incorporatingthe correct nucleotide, and during the incorporation event, thefluorophore switches on for a short time and switches off again. Theimaging occurs in real time at the time of the incorporation event. Atthe same time, incorporation events are happening at multiple attachmentelements in the sample container. Because the switching at these variousmolecules is stochastic, and not synchronized between the molecules,they can all be imaged at the same time as the reactions are occurring,so that a sequential and more time-consuming process is not required.

Before describing various super-resolution processes in detail, it isuseful to describe an example environment with which such processes canbe implemented. One such example environment is that of an imagescanning system, such as that illustrated in FIG. 1. The example imagescanning system may include a device for obtaining or producing an imageof a region. The example outlined in FIG. 1 shows an example imagingconfiguration of a backlight design.

As can be seen in the example of FIG. 1, subject samples are located onsample container 110, which is positioned on a sample stage 170 under anobjective lens 142. Light source 160 and associated optics direct a beamof light, such as laser light, to a chosen sample location on the samplecontainer 110. The sample fluoresces, and the resultant light iscollected by the objective lens 142 and directed to a photo-detectingcamera system 140 to detect the florescence. Sample stage 170 is movedrelative to objective lens 142 to position the next sample location onsample container 110 at the focal point of the objective lens 142.Movement of sample container 110 relative to objective lens 142 can beachieved by moving the sample stage itself, the objective lens, theentire optical stage, or any combination of the foregoing. Furtherexamples may also include moving the entire imaging system over astationary sample.

Fluid delivery module or device 100 directs the flow of reagents (e.g.,fluorescent nucleotides, buffers, enzymes, cleavage reagents, etc.) to(and through) sample container 110 and waste valve 120. In someapplications, the sample container 110 can be implemented as a flowcellthat includes clusters of nucleic acid sequences at a plurality ofsample locations on the sample container 110. The samples to besequenced may be attached to the substrate of the flowcell, along withother optional components.

The system also comprises temperature station actuator 130 andheater/cooler 135 that can optionally regulate the temperature ofconditions of the fluids within the sample container 110. Camera system140 can be included to monitor and track the sequencing of samplecontainer 110. Camera system 140 can be implemented, for example, as aCCD camera, which can interact with various filters within filterswitching assembly 145, objective lens 142, and focusing laser 150.Camera system 140 is not limited to a CCD camera, and other cameras andimage sensor technologies can be used.

Light source 160 (e.g., an excitation laser within an assemblyoptionally comprising multiple lasers) or other light source can beincluded to illuminate fluorescent sequencing reactions within thesamples via illumination through fiber optic interface 161 (which canoptionally comprise one or more re-imaging lenses, a fiber opticmounting, etc.). Low watt lamp 165, focusing laser 150, focusingdetector 141, and reverse dichroic 185 are also presented in the exampleshown. Focusing laser 150 may be used together with focusing detector141 to auto-focus the system, i.e., by adjusting the distance betweenobjective 142 and sample 110, or using other focusing techniques asknown in the art. In some applications focusing laser 150 may be turnedoff during imaging. In other applications, an alternative focusconfiguration can include a second focusing camera (not shown), whichcan be a quadrant detector, a Position Sensitive Detector (PSD), orsimilar detector to measure the location of the scattered beam reflectedfrom the surface concurrent with data collection.

Although illustrated as a backlit device, other examples may include alight from a laser or other light source (not shown) that is directedthrough the objective lens 142 onto the samples on sample container 110.Sample container 110 can be ultimately mounted on a sample stage 170 toprovide movement and alignment of the sample container 110 relative tothe objective lens 142. The sample stage can have one or more actuatorsto allow it to move in any of three directions. For example, in terms ofthe Cartesian coordinate system, actuators can be provided to allow thestage to move in the X, Y and Z directions relative to the objectivelens. This can allow one or more sample locations on sample container110 to be positioned in optical alignment with objective lens 142.

A focus (z-axis) component 175 is shown in this example as beingincluded to control positioning of the optical components relative tothe sample container 110 in the focus direction (typically referred toas the z axis, or z direction). Focus component 175 can include one ormore actuators physically coupled to the optical stage or the samplestage, or both, to move sample container 110 on sample stage 170relative to the optical components (e.g., the objective lens 142) toprovide proper focusing for the imaging operation. For example, theactuator may be physically coupled to the respective stage such as, forexample, by mechanical, magnetic, fluidic or other attachment or contactdirectly or indirectly to or with the stage. The one or more actuatorscan be configured to move the stage in the z-direction while maintainingthe sample stage in the same plane (e.g., maintaining a level orhorizontal attitude, perpendicular to the optical axis). The one or moreactuators can also be configured to tilt the stage. This can be done,for example, so that sample container 110 can be leveled dynamically toaccount for any slope in its surfaces.

Focusing of the system generally may refer to aligning the focal planeof the objective lens with the sample to be imaged at the chosen samplelocation. However, focusing can also refer to adjustments to the systemto obtain a desired characteristic for a representation of the samplesuch as, for example, a desired level of sharpness or contrast for animage of a test sample. Because the usable depth of field of the focalplane of the objective lens may be very small (sometimes on the order of1 μm or less), focus component 175 closely follows the surface beingimaged. Because the sample container is not perfectly flat as fixturedin the instrument, focus component 175 may be set up to follow thisprofile while moving along in the scanning direction (typically referredto as the y-axis herein).

The light emanating from a test sample at a sample location being imagedcan be directed to one or more detectors, including, for example, camerasystem 140. Detectors can include, for example a CCD camera. An aperturecan be included and positioned to allow only light emanating from thefocus area to pass to the detector. The aperture can be included toimprove image quality by filtering out components of the light thatemanate from areas that are outside of the focus area. Emission filterscan be included in filter switching assembly 145, which can be selectedto record a determined emission wavelength and to cut out any straylaser light.

In various examples, sample container 110 can include one or moresubstrates upon which the samples are provided. For example, in the caseof a system to analyze a large number of different nucleic acidsequences, sample container 110 can include one or more substrates onwhich nucleic acids to be sequenced are bound, attached or associated.In various examples, the substrate can include any inert substrate ormatrix to which nucleic acids can be attached, such as for example glasssurfaces, plastic surfaces, latex, dextran, polystyrene surfaces,polypropylene surfaces, polyacrylamide gels, gold surfaces, and siliconwafers. In some applications, the substrate is within a channel or otherarea at a plurality of locations formed in a matrix or array across thesample container 110.

One or more controllers (not illustrated) can be provided to control theoperation of a scanning system, such as the example scanning systemdescribed above with reference to FIG. 1. The controller can beimplemented to control aspects of system operation such as, for example,focusing, stage movement, and imaging operations. In variousapplications, the controller can be implemented using hardware,software, or a combination of the foregoing. For example, in someimplementations the controller can include one or more CPUs orprocessors with associated memory. As another example, the controllercan comprise hardware or other circuitry to control the operation. Forexample, this circuitry can include one or more of the following: fieldprogrammable gate array (FPGA), application specific integrated circuit(ASIC), programmable logic device (PLD), complex programmable logicdevice (CPLD), a programmable logic array (PLA), programmable arraylogic (PAL) or other similar processing device or circuitry. As yetanother example, the controller can comprise a combination of thiscircuitry with one or more processors.

Sequencing technologies that can be used with systems such as thatdescribed with reference to FIG. 1 include next-generation sequencing(NGS) technologies. Sequencing by Synthesis (SBS) is a widely adoptedNGS technology that uses modified dNTPs containing a terminator thatblocks further polymerization. The sequencing reaction may be conductedsimultaneously on a large number of template molecules. With SBS, afluorescently labeled reversible terminator is imaged as each dNTP isadded, and then cleaved to allow incorporation of the next base. Becauseall 4 reversible terminator-bound dNTPs are present during eachsequencing cycle, natural competition minimizes incorporation bias. Thisresults in a base-by-base sequencing that enables accurate data for abroad range of applications.

With NGS, DNA polymerase catalyzes the incorporation of fluorescentlylabeled deoxyribonucleotide triphosphates (dNTPs) into a DNA templatestrand during sequential cycles of DNA synthesis. During each cycle, atthe point of incorporation, the nucleotides are identified byfluorophore excitation. One difference is that, instead of sequencing asingle DNA fragment, NGS extends this process across millions offragments in a massively parallel fashion.

One approach to NGS includes four basic steps: library preparation,cluster generation, sequencing, and data analysis. With librarypreparation, the sequencing library is prepared by random fragmentationof the DNA or cDNA sample, followed by 5′ and 3′ adapter ligation.Alternatively, the fragmentation and ligation reactions are combinedinto a single step that greatly increases the efficiency of the librarypreparation process. Adapter-ligated fragments are then PCR amplifiedand gel purified.

For cluster generation, the library is loaded into a flow cell wherefragments are captured on a lawn of surface-bound oligos complementaryto the library adapters. Each fragment is then amplified into distinct,clonal clusters through bridge amplification. When cluster generation iscomplete, the templates are ready for sequencing. One approach to SBSuses a reversible terminator-based method that detects single bases asthey are incorporated into DNA template strands. As all four reversibleterminator-bound dNTPs are present during each sequencing cycle, naturalcompetition minimizes incorporation bias and reduces raw error ratescompared to other technologies. During data analysis and alignment, thenewly identified sequence reads are aligned to a reference genome.Following alignment, many variations of analysis are possible, such assingle nucleotide polymorphism (SNP) or insertion-deletion (indel)identification, read counting for RNA methods, phylogenetic ormetagenomic analysis, and more.

Important in imaging systems is the speed at which scanning operationscan take place. Consider sequencing systems, for example. In suchsystems, it is often desirable to increase the speed with which samplemolecules can be read. One way to increase the throughput of imagingsystems is to decrease the size and spacing of the structures beingimaged. In sequencing systems, this can be accomplished by packingtemplate molecules closer together to increase the number of reads thatcan be accomplished for a given unit area. However, resolution of theimaging system is limited by the wavelength of light, aperture of theoptics, and other factors.

In 1873 a German physicist by the name of Ernest Abbe published aformula defining the resolution limit of the microscope. Abbe's limit isdefined as:

$d = {\frac{\lambda}{2\; {NA}}.}$

Where λ is the wavelength of the light waves illuminating the specimen,or the excitation wavelength band in fluorescence. NA is the numericalaperture of the objective lens, which is defined by the refractive indexof the transmission medium, n, multiplied by the sine of the apertureangle (sin(α)), where α=half-angle of maximum cone of light that canenter or exit the lens. Accordingly, NA can be set forth as NA=n·sin(α),and Abbe's Limit can be rewritten as:

$d = {\frac{\lambda}{2\; n\; \sin \; \alpha}.}$

This resolution limit, often referred to as the diffraction barrier,defines the ability of the optical instrument to distinguish between twoobjects separated by a lateral distance less than approximately half thewavelength of light used to image the specimen. The 2014 Nobel Prize forchemistry was awarded for bypassing this scientific limitation. Indeed,Abbe's limit has now been overcome by a number of techniques. Theseinclude: STochastic Optical Reconstruction Microscopy (STORM),STimulated Emission Depletion Microscopy (STED), PhotoActivationLocalization Microscopy (PALM) and Structured Illumination Microscopy(SIM). All of these methods allow resolutions to be achieved of muchlower than 200 nm, down to ˜20 nm for STORM, STED and PALM, and about100 nm for SIM.

STORM, for example, relies on stochastic switching of single-moleculefluorescence so that only a small fraction of the fluorophores isactivated stochastically at any given time. The activated fluorophoresare separated sufficiently such that they can be resolved within Abbe'slimit. This enables determining their positions with sufficientprecision. However, often it involves the process be repeated andmultiple images (snapshots) of the sample be taken, each capturing arandom subset of the fluorophores, so that a final image can bereconstructed. The final image is generated by accumulating the multipleimages. Accordingly, the activations are physically separated so thatthey can be optically resolved.

Although the systems and methods may be described herein from time totime in the context of this example system of FIG. 1, this is only oneexample with which these systems and methods might be implemented. Thesystems and methods described herein can be implemented with this andother scanners, microscopes and other imaging systems.

The sample container for an imaging system may be constructed as anarray of single-molecule attachment elements. An example container forsingle molecule stochastic sequencing is illustrated in FIG. 2. Such acontainer, with multiple rows of attachment elements 212 can allowseveral DNA molecules to be individually sequenced in parallel. However,the minimum spacing, d, of the attachment elements 212 in variousapplications can be limited by the optical resolution of the system. Insome applications, the attachment elements 212 can include zero-modewaveguides, which are optical nanostructures that serve to confine theobservation volume, thereby extending the concentrations forsingle-molecule microscopy.

FIG. 3 illustrates an example of a side view of a row of the containerillustrated in FIG. 2. FIG. 4 illustrates an example process forsequencing. At operation 410, the sample container is fabricated andprovided with attachment elements, or anchors (e.g., attachment elements212). The attachment elements are provided having a pitch, d. In someapplications, the pitch, d, can be the same in both directions, while inother applications it can vary. For example, the pitch, d, in someapplications can be less than about 20 nm. As another example, in someapplications the pitch can range in dimension from about 2 nm to about20 nm. In some applications the pitch can be greater than or less thanthis range of dimensions. In further examples, the pitch can be reducedto the smallest dimension possible without the individual moleculesbeing affected by physical interactions between them such as, forexample, charge-charge interactions between the molecules.

In this example, single-pot real-time chemistry is used for sequencing.Individual DNA template molecules 310 are provided. These may beanchored to the attachment elements 212 (e.g., one per attachmentelement 212) as shown in FIG. 3 at 302. Accordingly, at operation 412, asingle DNA template molecule 310 is attached to each attachment element.In one example, to ensure that only one molecule exists per patternedattachment point 212, the attachment points may be fabricated at a verysmall scale, approaching the molecular scale (>˜20 nm). In this way,through steric hindrance this can help to ensure that only one DNAmolecule attaches at each location. Sequencing single molecules canallow a high density of clusters, as the clusters can be the smallestpossible size. This allows the effect of cluster size to be removed as afactor for determining the minimum pitch. Also, with single-moleculesequencing, there is little or no risk of “pad-hopping”, whereby acluster grows across the interstitial space and creates a neighboringduplicate. This hopping is a consequence of the amplification processand generally will not occur when there is no amplification. Also,because there is no amplification, this may also save not only time, butalso the cost of the reagents required. By performing single-moleculesequencing, it is then possible to account for phasing errors in eachmolecule during sequencing.

At operations 414 and 416, a sequencing primer is attached andsingle-pot reagents are added. This is shown in FIG. 3 at 304. Thesereagents 314 can include a set of four nucleotides that have a 5′diphosphate quencher molecule and 3′ phosphate block, with a labelmoiety (e.g., dye molecule).

As used herein, the term “nucleic acid” can be used refer to at leasttwo nucleotide analog monomers linked together. A nucleic acid cancontain phosphodiester bonds, however, in some applications, a nucleicacid can be an analog having other types of backbones, comprising, forexample, phosphoramide, phosphorothioate, phosphorodithioate, peptidenucleic acid backbones and linkages, positive backbones, or non-ionicbackbones. A nucleic acid can include a pentose moiety such as ribose(present in naturally occurring RNA), deoxy-ribose (present in naturallyoccurring DNA) or dideoxy ribose. In some applications a nucleic acidcan have a non-pentose moiety or carbocyclic sugar instead of a riboseor deoxyribose moiety. A nucleic acid can have one or more differentbase moieties including, but not limited to, adenine (A), guanine (G),thymine (T), uracil (U), cytosine (C), inosine, xanthanine,hypoxanthanine, isocytosine, isoguanine, nitropyrrole (including3-nitropyrrole) and/or nitroindole (including 5-nitroindole). Nucleicacids may be single stranded or double stranded, as specified, orcontain portions of both double stranded and single stranded sequence.The nucleic acid may be DNA (e.g. genomic DNA or cDNA), RNA or a hybrid.

As used herein, the term “nucleotide analog” is intended to includenatural nucleotides, non-natural nucleotides, ribonucleotides,deoxyribonucleotides, dideoxyribonucleotides and other molecules knownas nucleotides. The term can be used to refer to a monomer unit that ispresent in a polymer, for example to identify a subunit present in a DNAor RNA strand. The term can also be used to refer to a monomericmolecule that is not necessarily present in a polymer, for example, amolecule that is capable of being incorporated into a polynucleotide ina template dependent manner by a polymerase. The term can refer to anucleoside unit having, for example, 0, 1, 2, 3, 4, 5 or more phosphateson the 5′ carbon. A nucleotide analog can have a base moiety including,but not limited to, adenine (A), guanine (G), thymine (T), uracil (U),cytosine (C), inosine, xanthanine, hypoxanthanine, isocytosine,isoguanine, nitropyrrole (including 3-nitropyrrole) and/or nitroindole(including 5-nitroindole). Example natural nucleotides include, withoutlimitation, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP,dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, anddGMP.

Non-natural nucleotides include those that are not present in a naturalbiological system. A non-natural nucleotide can be incapable of beingfurther extended after being incorporated into a polynucleotide.Examples include, nucleotide analogs having a reversible ornon-reversible blocking moiety. A natural or non-natural nucleotide canbe capable of being further extended after being incorporated into apolynucleotide. Examples include, nucleotide analogs having a 3′hydroxyl. In some applications, the nucleotide analog(s) will notinclude a reversible blocking moiety, or the nucleotide analog(s) willnot include a non-reversible blocking moiety or the nucleotide analog(s)will not include any blocking moiety at all.

As used herein, the term “blocking moiety,” when used in reference to anucleotide analog, means a part of the nucleotide analog that inhibitsor prevents the nucleotide analog from forming a covalent linkage to asecond nucleotide analog. For example, in the case of nucleotide analogshaving a pentose moiety, a blocking moiety can prevent formation of aphosphodiester bond between the 3′ oxygen of the nucleotide and the 5′phosphate of the second nucleotide. The blocking moiety can be part of anucleotide that is a monomer unit present in a nucleic acid polymer orthe blocking moiety can be a part of a free nucleotide analog (e.g. anucleotide triphosphate). The blocking moiety that is part of anucleotide analog can be reversible, such that the blocking moiety canbe modified to render the nucleotide analog capable of forming acovalent linkage to a second nucleotide analog. Particularly usefulreversible blocking moieties are phosphates, phosphodiesters,phosphotriesters, phosphorothioate esters, and carbon esters. Furtherexamples of reversible blocking moieties that can be used are set forthbelow and in references incorporated by reference herein as set forthbelow. In particular applications, a blocking moiety, such as areversible blocking moiety, can be attached to the 3′ position or 2′position of a pentose moiety of a nucleotide analog.

As used herein, the term “label moiety,” when used in reference to anucleotide analog, means a part of the nucleotide analog that provides adistinguishable characteristic that is not otherwise manifest in thenucleotide analog. The distinguishable characteristic can be, forexample, an optical signal such as absorbance of radiation, fluorescenceemission, luminescence emission, fluorescence lifetime, fluorescencepolarization, or the like; binding affinity for a ligand or receptor;magnetic properties; electrical properties; charge; mass; radioactivityor the like. Example label moieties include, without limitation, afluorophore, luminophore, chromophore, radioactive isotope, mass label,charge label, spin label, receptor, ligand, or the like. The labelmoiety can be part of a nucleotide that is a monomer unit present in anucleic acid polymer or the label moiety can be a part of a freenucleotide analog (e.g. a nucleotide triphosphate).

As used herein, the term “label-modifier moiety,” when used in referenceto a nucleotide analog having a label moiety, means a part of thenucleotide analog that changes a distinguishable characteristic of thelabel moiety. Typically, the change in the distinguishablecharacteristic is manifest in the presence of the label-modifier moietybut not in the absence of the label-modifier moiety. For example, alabel-modifier moiety can be a quencher that reduces fluorescence orluminescence emission from a label. In another example, a label-modifiermoiety can be a Förster resonance energy transfer (FRET) donor oracceptor that changes the intensity or wavelength of fluorescence orluminescence emission detected from the label. The label-modifier moietycan be part of a nucleotide that is a monomer unit present in a nucleicacid polymer or the label-modifier moiety can be a part of a monomericnucleotide analog (e.g. a nucleotide triphosphate).

As used herein, the term “deblocking agent” means a catalyst, enzyme,reagent or other substance that is capable of modifying or removing ablocking moiety. In particular applications, a deblocking agent can havespecificity for a blocking moiety that is part of a nucleotide that is amonomer unit present in a nucleic acid polymer. As such the deblockingagent may selectively remove a blocking moiety from a nucleotide analogthat is present in a nucleic acid compared to a blocking moiety that ispart of a monomeric nucleotide analog (e.g. a nucleotide triphosphate).Alternatively or additionally, a deblocking agent can selectively removea blocking moiety from a nucleotide analog that is present in a doublestranded nucleic acid compared to a blocking moiety that is part of amonomeric nucleotide analog (e.g. a nucleotide triphosphate) or part ofa nucleotide analog that is a monomer in a single stranded nucleic acid.Accordingly, in some applications the deblocking agent can have littleor no ability to remove a blocking moiety from a monomeric nucleotideanalog (e.g. a nucleotide triphosphate) or from nucleotide analog thatis a monomer in a single stranded nucleic acid. Example deblockingagents include, but are not limited to, an enzyme, such as aphosphoesterase, phosphodiesterase, phosphotriesterase, esterase, alkyltransferase or methyl transferase; or a chemical reagent.

As used herein, reference to “selectively” manipulating (e.g., to“selectively” remove) a first thing compared to second thing is intendedto mean that the manipulation has a greater effect on the first thingcompared to the effect on the second thing. The manipulation need nothave an effect on the second thing. The manipulation can have an effecton the first thing that is at least 1%, 5%, 10%, 25%, 50%, 75%, 90%,95%, or 99% greater than the effect on the second thing. Themanipulation can have an effect on the first thing that is at least 2fold, 3 fold, 4 fold, 5 fold, 10 fold, 100 fold, 1×10³ fold, 1×10⁴ foldor 1×10⁶ fold higher than the effect on the second thing. Themanipulation can include, for example, modifying, contacting, treating,changing, cleaving (e.g. of a chemical bond), photo-chemically cleaving(e.g. of a chemical bond), forming (e.g. of a chemical bond),photo-chemically forming (e.g. of a chemical bond), covalentlymodifying, non-covalently modifying, destroying, photo-ablating,removing, synthesizing, polymerizing, photo-polymerizing, amplifying(e.g. of a nucleic acid), copying (e.g. of a nucleic acid), extending(e.g. of a nucleic acid), ligating (e.g. of a nucleic acid), or othermanipulation set forth herein or otherwise known in the art. As usedherein, the term “transient,” when used in reference to a species in areaction or reaction cycle, means the species is present onlytemporarily during the course of the reaction or reaction cycle. Atransient species can be present, for example, for a time period that isno more than about 10 minutes, 1 minute, 30 seconds, 10 seconds, 1second, 100 milliseconds, 10 milliseconds, 1 millisecond, 100nanoseconds, 10 nanoseconds, or 1 nanosecond. In particularapplications, the transient species is present for a temporary timeperiod that is sufficient to allow detection of the transient species.For example, additionally or alternatively to the example maximum timesperiods set forth above, a transient species may be present for at least1 minute, 30 seconds, 10 seconds, 1 second, 100 milliseconds, 10milliseconds, 1 millisecond, 100 nanoseconds, 10 nanoseconds, 1nanosecond or 1 picosecond.

As used herein, the term “reaction cycle,” when used in reference to areactant and product, means a sequence of two or more reactions thatconvert the reactant to at least one transient species and then convertthe at least one transient species to the product. The reaction cyclecan be repeated, for example, such that the product serves as a reactantin the same sequence of reactions. For example, a nucleic acid primercan be extended by a single nucleotide in a first reaction cycle toproduce a primer extension product (having a single nucleotide added tothe original primer) and then the primer extension product can beextended again in a second reaction cycle to produce a primer extensionproduct (having two nucleotides added to the original primer). Therepetition of the cycle can use slightly different reactants, forexample, different nucleotide analogs can be added in sequential cyclesof primer extension. However, a reaction cycle need not be repeated. Anucleic acid reaction cycle can, for example, result in the addition ofa single nucleotide to a primer (e.g. in a polymerase catalyzedreaction) or in the addition of a single oligonucleotide to a primer(e.g. in a ligase catalyzed reaction).

Labels that are optically detectable are particularly useful. Examplesinclude chromophores, luminophores and fluorophores. Fluorophores areparticularly useful and include, for example, fluorescent nanocrystals;quantum dots, fluorescein, rhodamine, tetramethylrhodamine, eosin,erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, Cy3,Cy5, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, Alexa dyes, SETAdyes, Atto dyes, phycoerythin, bodipy, and analogs thereof. Usefuloptical probes are described in Haugland, Molecular Probes Handbook,(Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.),Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum PressNew York (1999), or WO 98/59066; WO 91/06678 or US Pat. Appl. Publ. No.2010/0092957 A1, each of which is incorporated herein by reference.Optical labels provide an advantage of rapid, relatively non-invasivedetection thereby allowing real time monitoring of a cyclic reaction.

Other labels, some of which are non-optical labels, can be used invarious applications of the methods and compositions set forth herein.Examples include, without limitation, an isotopic label such as anaturally non-abundant radioactive or heavy isotope; magnetic substance;electron-rich material such as a metal; electrochemiluminescent labelsuch as Ru(bpy)32+; or moiety that can be detected based on a nuclearmagnetic, paramagnetic, electrical, charge to mass, or thermalcharacteristic. Labels can also include magnetic particles or opticallyencoded nanoparticles. Such labels can be detected using appropriatemethods known to those skilled in the art. For example, a charged labelcan be detected using an electrical detector such as those used incommercially available sequencing systems from Ion Torrent (Guilford,Conn., a Life Technologies subsidiary) or detection systems described inUS Pat. App. Publ. Nos. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143A1; and 2010/0282617 A1, each of which is incorporated herein byreference. It will be understood that for some applications a nucleotideanalog need not have a label.

Another type of label that can be useful is a secondary label that isindirectly detected, for example, via interaction with a primary label,binding to a receptor or conversion to a detectable product by an enzymecatalyst or other substance. An example secondary label is a ligand suchas biotin or analogs thereof that can be detected via binding to areceptor such as avidin, streptavidin or analogs thereof. Other usefulligands are epitopes that can bind to receptors such as antibodies oractive fragments thereof, and carbohydrates that can bind to receptorssuch as lectins. The receptors can be labeled, for example, with anoptical label, to allow them to be detected. In particular applications,the ligand can be attached to a nucleotide analog in a way that reducesor prevents affinity to a receptor. Release of the ligand can then bedetected based on affinity of the ligand for its respective receptorwhen detached from the nucleotide analog. The ligand can further beattached to a blocking moiety or may itself function as a blockingmoiety, as set forth above more generally for label moieties. Thus,removal of the ligand from a nucleotide analog can function to deblockthe nucleotide analog and to provide a detectable event.

Another example secondary label is pyrophosphate or analogs thereof.Pyrophosphate can be detected by solid-phase chelators and/or electronicbiosensors. In some applications, pyrophosphate can be detected by acascade of enzymes that converts pyrophosphate to ATP and then tochemiluminescence. Example enzyme cascades include those typically usedin pyrosequencing and/or described in US Pat App. Publ. No. 2005/0244870A1, which is incorporated herein by reference. In some applications, useof an enzyme cascade detection system that produces ATP may require useof an Adenine nucleotide analog, such as ATPv̧S, that is incorporatedinto a primer by polymerase but does not cause a background signal thatcompetes with the pyrophosphate signal. In particular applications,pyrophosphate or an analog thereof can be attached to a nucleotideanalog at a position other than the 5′ position where a triphosphateresides. This nucleotide analog can produce two pyrophosphate-inducedsignals in an appropriate detection system, one due to the release ofpyrophosphate from the 5′ position (due to polymerase activity) and asecond due to release from the other position, for example, by adeblocking agent. Production of two pyrophosphate-induced signals canprovide an advantage of increased signal to noise in a detection step orincreased accuracy in evaluating sequencing data. A particularly usefulanalog of pyrophosphate, when present on a nucleotide analog, will becharge-neutral at one or more of the oxygen moieties that are typicallynegatively charged in pyrophosphate. In one example the pyrophosphateanalog can have no charged oxygen atoms. Charge neutrality may favorinteractions with some polymerase species. The pyrophosphate analog,once released, can be converted to a form for interaction with enzymesin a detection cascade if appropriate or otherwise desired.

A label moiety that is used in a method or composition set forth hereincan be an intrinsic label (i.e. an endogenous label) that is present ina naturally occurring molecule being detected, such as a proton orpyrophosphate that is released from a nucleotide analog uponincorporation into an extended primer. Pyrophosphate release can bedetected using a pyrosequencing or similar technique, examples of whichare commercially available from 454 Life Sciences (Branford, Conn., aRoche Company) or described in US Pat App. Publ. No. 2005/0244870 A1,which is incorporated herein by reference. Example systems for detectingprimer extension based on proton release include those that arecommercially available from Ion Torrent (Guilford, Conn., a LifeTechnologies subsidiary) or described in US Pat. App. Publ. Nos.2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; and 2010/0282617 A1,each of which is incorporated herein by reference. Alternatively oradditionally to detection of an intrinsic label, one can detect a labelthat is exogenous to a natural nucleotide analog. Thus, in someapplications solely exogenous probes are detected such that endogenousprobes are not detected, in other applications solely endogenous probesare detected such that exogenous probes are not detected and in someapplications a combination of exogenous and endogenous probes aredetected.

In some applications a label moiety that is detectable under theconditions being used is not necessary or not desirable. Thus, anucleotide analog that is present in a reaction mixture or used in areaction set forth herein may lack a particular detectable label moietywhen in a monomeric form and when incorporated into an extended primer.The nucleotide analog may nonetheless include a blocking moiety. In suchapplications, detection may not be carried out at all.

In addition to a label moiety, a nucleotide analog can further include alabel-modifier moiety. A label-modifier moiety can function to modify asignal produced by the label moiety. In some applications, a signal thatis produced by the label moiety in the presence of the label-modifiermoiety can be distinguished from a signal that is produced by the labelmoiety in the absence of the label-modifier moiety. For example, thelabel moiety and label-modifier moiety can be a Förster resonance energytransfer (FRET) donor-acceptor pair. As such, a change in the wavelengthof apparent fluorescence emission from a nucleotide analog can bedetected and will be indicative of the presence or absence of thelabel-modifier moiety. Example fluorophores that can be used as membersof FRET pairs include, but are not limited to, fluorescent nanocrystals;quantum dots; d-Rhodamine acceptor dyes including dichloro[R110],dichloro[R6G], dichloro[TAMRA], dichloro[ROX] or the like; fluoresceindonor dye including fluorescein, 6-FAM, or the like; Cyanine dyes suchas Cy3B; Alexa dyes, SETA dyes, Atto dyes such as Atto 647N which formsa FRET pair with Cy3B and the like.

In another example, the intensity of a signal from a label moiety thatoccurs in the presence of the label-modifier moiety can be distinguishedfrom the intensity of signal that is produced in the absence of thelabel-modifier moiety. For example, the label can be a fluorophore andthe label-modifier moiety can be a quencher such that absence of thelabel-modifier moiety can be detected as an apparent increase offluorescence emission from the nucleotide analog. Example quenchersinclude, but are not limited to,DACYL(4-(4′-dimethylaminophenylazo)benzoic acid), Black Hole Quenchers(Biosearch Technologies, Novato, Calif.), QxI quenchers (Anaspec,Freemont, Calif.), Iowa black quenchers, DABCYL, BHQ1, BHQ2, QSY7, QSY9,QSY21, QSY35, BHQO, BHQ1, BHQ2, QXL680, ATTO540Q, ATTO580Q, ATTO612Q,DYQ660, DYQ661 and IR Dye QC-1 quenchers.

An example of the ratchet biochemistry components is shown in FIG. 5.This example includes a 3′ phosphate dye and a 5′ tri-phosphatequencher. This example kit also includes a polymerase that canincorporate the nucleotides, and also a dsDNA specific enzyme that willcleave the 3′ phosphate block but only from nucleotides that have beenincorporated.

Any of a variety of polymerases can be used in a method or compositionset forth herein including, for example, protein-based enzymes isolatedfrom biological systems and functional variants thereof. Reference to aparticular polymerase, such as those exemplified below, will beunderstood to include functional variants thereof unless indicatedotherwise. A particularly useful function of a polymerase is to catalyzethe polymerization of a nucleic acid strand using an existing nucleicacid as a template. Other functions that are useful are describedelsewhere herein. Examples of useful polymerases include DNA polymerasesand RNA polymerases. Particularly useful polymerases include Pol217 andPol427 as set forth in the Examples section below and other polymerasedescribed in US 2006/0240439 A1, which is incorporated herein byreference.

A polymerase having an intrinsic 3′ to 5′ proofreading exonucleaseactivity can be useful for some applications. Polymerases thatsubstantially lack 3′ to 5′ proofreading exonuclease activity are alsouseful in some applications, for example, in most sequencingapplications. Absence of exonuclease activity can be a wild typecharacteristic or a characteristic imparted by a variant or engineeredpolymerase structure. For example, exo minus Klenow fragment is amutated version of Klenow fragment that lacks 3′ to 5′ proofreadingexonuclease activity.

Polymerases can be characterized according to their rate of dissociationfrom nucleic acids. In particular applications it is desirable to use apolymerase that has a relatively high dissociation rate. This can beuseful for example, in applications where dissociation of the polymeraseallows a deblocking step to proceed. For example, an enzyme when used asa deblocking agent may be sterically blocked by a polymerase such thatthe enzyme is prevented from removing a blocking moiety from an extendedprimer. In such a case, the lifetime of the extended primer having theblocking moiety can be influenced by the dissociation rate of thepolymerase. The dissociation rate is an activity of a polymerase thatcan be adjusted to tune reaction rates in methods set forth herein.

Depending on the example that is to be used, a polymerase can be eitherthermophilic or heat inactivated. Thermophilic polymerases are typicallyuseful for high temperature conditions or in thermocycling conditionssuch as those employed for polymerase chain reaction (PCR) techniques.Examples of thermophilic polymerases include, but are not limited to 9°N DNA Polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNApolymerase, RB69 DNA polymerase, KOD DNA polymerase, and VentR® DNApolymerase. Most polymerases isolated from non-thermophilic organismsare heat inactivated. Examples are DNA polymerases from phage. It willbe understood that polymerases from any of a variety of sources can bemodified to increase or decrease their tolerance to high temperatureconditions.

FIG. 6 illustrates an example of a chemical process upon theincorporation of the nucleotides. Referring now to FIG. 6, at 614, as anucleotide is incorporated the 5′ triphosphate is cleaved. Because theQuencher molecule is attached, this Quencher molecule is also cleaved.As a result, the fluorescent dye is no longer quenched and can emitfluorescence as shown in 616. At 618, now that the nucleotide isincorporated in dsDNA the second enzyme is then able to cleave the 3′phosphate, along with the attached dye. This is shown in FIG. 3 at 306.This renders the molecule dark again as shown at 620, and generates anew 3′ OH, ready for the next incorporation (shown at 622). There issome time that passes between the incorporation event occurring and thefluorophore switching on, and the removal of phosphate block causing thefluorophore to diffuse away (out of the excitation volume) and switchingoff.

As another further example, in some applications, the sequencing processcan include providing a mixture including an enzyme capable of couplingnucleotides, a deblocking agent, a nucleic acid bound to a strand ofnucleotides having a sequence complimentary to the nucleotide sequencesanchored to the solid support, and more than one nucleotide analogincluding a base with a label moiety and corresponding quencher moietybound thereto. The label moieties may be correlated with a specific basemoiety. The process may further allow sequential addition of a pluralityof the nucleotide analogs to the nucleic acid to proceed via severalreaction cycles in the mixture while concurrently imaging (e.g.,operation 418, below) the label moieties within the array. In someapplications, each reaction cycle may include: (i) the polymerase addinga nucleotide analog to the nucleic acid by cleaving the quencher moietyand forming a transient nucleic acid species comprising the labelmoiety; and (ii) the deblocking agent modifying the transient nucleicacid species to remove the label moiety. In various applications, theseveral reaction cycles may include at least 100 reaction cycles,whereby the nucleic acid is extended by addition of at least 100nucleotide analogs. The enzyme capable of coupling nucleotides mayinclude a polymerase, a myosin or a kinase. The nucleotide analog mayinclude a pentose moiety having a 3′ carbon and the label moiety may beattached to the nucleotide at the 3′ carbon. In another example, thenucleotide analog may include a triphosphate moiety and the quenchermoiety may be attached to the triphosphate moiety.

In some applications, the deblocking agent may include a phosphoesteraseenzyme (e.g., phosphodiesterase, phosphotriesterase), which may beincluded to selectively remove a phosphodiester moiety or thephosphotriester moiety from the transient nucleic acid species. Thephosphoesterase may be selected from the group consisting ofEndonuclease IV and AP endonuclease. The transient nucleic acid speciesmay be present for at least 1 millisecond before the deblocking agentmodifies the transient nucleic acid species to remove the label moiety.As a further example, the transient nucleic acid species may be presentfor no more than 30 seconds before the deblocking agent modifies thetransient nucleic acid species to remove the label moiety.

Returning now to FIG. 4, at operation 418 an imaging system of thesequencer detects the intensity of the signal in each of the fourchannels (ACGT), and the fluorescence of the dye is manifested as anincrease in signal corresponding to the base that has been incorporated.Accordingly, the fluorescence is detected by an image sensor in theimaging system (e.g., camera system 140 in the example imaging system ofFIG. 1), and the images can be recorded. The imaging and recordingsystem can image and record multiple channels at the same time (e.g.,one channel for each base), and each channel can image and record the onand off sequences for that channel at all of the locations within itsfield of view. Because of the stochastic nature of the process, all ofthe molecules within the field of view of the imaging system can beactivated at the same time and the reactions occurring recorded in eachchannel simultaneously for all of the molecules within the field ofview.

In other words, in various applications the sequencing is occurring byincorporating the correct nucleotides by polymerase for a givenmolecule, while other incorporation events are going on around itrandomly and the imaging can be running in real time to detect thereactions in each of the molecules as they are occurring. As noted, theimaging can be arranged to observe the four different color channels,one for each base, and each channel can detect and record itsflorescence turning on and off.

FIG. 7 illustrates an example of the process of super-resolution imagingusing the above-described chemical process. This shows an example of the‘on’ and ‘off’ events 742 as nucleotide incorporation and deblockingevents occur over time for a single molecule 722. The time traces of theon/off state for each molecule to determine the sequence of thatmolecule. In the illustrated example, ‘on’ states 744 are illustratedfor molecule 722 in a sequence of ACTGCT.

In various applications, the incorporation and deblocking events arestochastic and not synchronized between molecules. Therefore,statistically, ‘on’ events for a given base for a given molecule at agiven location may occur at different times from ‘on’ events for thatbase for other molecules at adjacent or surrounding locations.Accordingly, for each channel, there is a statistical probability thatfor each channel an on-events for that channel are sufficientlyspatio-temporally separated such that they are resolvable as separateevents by the imaging system despite the fact that the molecules arespaced at a pitch smaller than would otherwise be allowed by Abbe'slimit. This randomly generated spatio-temporal separation between ‘on’events for a given base provides a greater effective pitch betweenevents than the actual well spacing, making the ‘on’ events resolvableby the optics of the imaging system. An example of this is shown at 304in FIG. 3. In this example, each of the molecules in this center row iscurrently exhibiting and on an event for a base that is different fromits adjacent molecules. In this example, the molecules from left toright are exhibiting on events for the bases A, C, G, T, G, A, C, T andC. The nearest occurrences of an on event for the same bases are the twoC events on the right-hand side of the road, and the 2 G events towardthe center of the row. In each case, these are spaced 2 d, or twice theaverage pitch of the attachment elements 212.

Because the photo-switching is stochastic, there may be occasions whentwo or more molecules closer together than otherwise allowed by Abbes'limit (e.g., 2 adjacent molecules) are switched on for the same base atthe same time. In this case, these molecules might not be resolved. Thispossibility can be mitigated by controlling the switching rates torender these coincident adjacent events to be rare. For example, theconcentrations of nucleotides and enzyme can be adjusted to allow thedyes to remain ‘on’ for a sufficient time to identify the base. Also,these concentrations can be selected so that not only is the ‘on’ timesufficient, but also so the ‘off’ time is long enough so that there arefew or no similar ‘on’ events occurring in close proximity to oneanother. For example, in one application, the on and off times areselected so that the probability of a dye being on for a given locationat the same time a dye is on at an immediately adjacent location is lessthan or equal to 0.5%. In another application, the on and off times areselected so that the probability of a dye being on for a given locationat the same time a dye is on at an immediately adjacent location isgreater than or equal to 0.5%. In other applications, the on and offtimes are selected so that the probability of a dye being on for a givenlocation at the same time a dye is on at an immediately adjacentlocation is in the range of 0.1% to 0.5%. In other applications, the onand off times are selected so that the probability of a dye being on fora given location at the same time a dye is on at an immediately adjacentlocation is in the range of 0.1% to 0.8%. In another application, the onand off times are selected so that this adjacency probability is lowerthan an acceptable error rate in the given sequencing application inwhich it is applied.

There may also be other ways to address the situation in which ‘on’events for a given channel occur too close together to be resolved bythe imaging system. In one example, the system can detect anillumination intensity greater than an average or baseline illuminationintensity, or other threshold, indicating that illumination eventsoccurred for a given base for two or more molecules closer together thanAbbe's limit. Likewise, the spot size or spot shape may also be used todetermine a situation in which two or more molecules closer togetherthan Abbe's limit are exhibiting and on event at the same time for agiven base. Accordingly, comparing an illumination event to anillumination intensity threshold, spot size threshold, or both, can be atechnique used to allow the system to determine whether two adjacentmolecules exhibited an on event at the same time for the same base. Thismay allow for increased tolerance of the system to these adjacencies,which may in turn allow the on and off times to be selected to permit ahigher adjacency probability than might otherwise be tolerated withouteither or both of these threshold determinations. Further examples mayalso be implemented such that once aligned to a reference genome,apparent “deletion” errors may be resolved by observing which basesoccurred in adjacent reads at that same moment in time. For example adeletion that should have been a “C” coincides with a correctly called“C” in an adjacent site.

Processes described herein provide a way to achieve photo-switching, byenzymatic cycling of fluorophores between dark and light states. Thiscycling could be through one of many enzymes, e.g. polymerase, such asthose described herein. It could also be through other enzymes that turnover nucleotides (e.g. Adenosine triphosphate (ATP) by myosins, orkinesins), or any multi-partite substrate in which the enzymatic processseparates the parts (e.g. a quencher from a fluorescent dye). Thesecould be quenched at the 5′ end (with a 3′-dye label) similar to theprocess described above, and therefore when they are hydrolyzed theyfluoresce, and then return to darkness when the ADP products arereleased. Another example can be an enzymatic processes that joinsnon-fluorescent molecules together in a reaction that yields afunctional fluorophore. This fluorophore may then get broken down againby application of an orthogonal chemistry.

In further applications, the systems and methods described herein canuse alternative techniques to the above-described ratchet chemistryprocess to achieve stochastic photo-switching of molecules insimultaneous adjacent reactions. For example, the photo-switchingtechniques of STORM (STochastic Optical Reconstruction Microscopy) orDNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT)techniques for photo-switching of the fluorophores can be used asdescribed above, with similar effects as achieved by the chemistrydescribed above. Accordingly, the on and off switching may be achievedin a number of different ways, such as, for example, direct switchingdriven by photochemical reactions (dSTORM), or transient DNAhybridization of DNA labels (DNA-PAINT). Various reagents could becreated, with e.g. antibodies against specific targets, coupled to suchenzymatic moieties for driving the photo-switching.

STORM, for example, originally overcame Abbe's limit by havingfluorophores that switch on and off in spatially remote locations, andsequential frames of these events are recorded as the locations change.In any given frame, only a small fraction of the fluorophores would beswitched on, and these are sufficiently separated beyond Abbe's limit.However, this is a sequential process in which the fluorophores areenergized, recorded and erased and the process then must be repeated forall locations until all molecules are captured. This would require theacquisition of many successive frames so that all molecules can berecorded and a complete image of the object obtained. However,applications of the processes described herein enable the reactions forall of the molecules within the field of view to occur at the same time(instead of energizing, erasing and repeating for sufficiently separatedmolecules) and imaging the process in real-time as it occurs. This takesadvantage of the stochastic nature of the reactions so that they arestatistically not occurring next to one another frequently and they canbe captured by the multiple channels (e.g., one for each base) of theimaging system in real-time.

While various examples of the disclosed technology have been describedabove, it should be understood that they have been presented by way ofexample only, and not of limitation. Likewise, the various diagrams maydepict an example architectural or other configuration for the disclosedtechnology, which is done to aid in understanding the features andfunctionality that can be included in the disclosed technology. Thedisclosed technology is not restricted to the illustrated examplearchitectures or configurations, but the desired features can beimplemented using a variety of alternative architectures andconfigurations. Indeed, it will be apparent to one of skill in the arthow alternative functional, logical or physical partitioning andconfigurations can be implemented to implement the desired features ofthe technology disclosed herein. Also, a multitude of differentconstituent module names other than those depicted herein can be appliedto the various partitions. Additionally, with regard to flow diagrams,operational descriptions and method claims, the order in which the stepsare presented herein shall not mandate that various examples beimplemented to perform the recited functionality in the same orderunless the context dictates otherwise.

Although the disclosed technology is described above in terms of variousexample examples and implementations, it should be understood that thevarious features, aspects and functionality described in one or more ofthe individual examples are not limited in their applicability to theparticular example with which they are described, but instead can beapplied, alone or in various combinations, to one or more of the otherexamples of the disclosed technology, whether or not such examples aredescribed and whether or not such features are presented as being a partof a described example. Thus, the breadth and scope of the technologydisclosed herein should not be limited by any of the above-describedexample examples. It should be appreciated that all combinations of theforegoing concepts (provided such concepts are not mutuallyinconsistent) are contemplated as being part of the inventive subjectmatter disclosed herein. In particular, all combinations of claimedsubject matter appearing at the end of this disclosure are contemplatedas being part of the inventive subject matter disclosed herein.

Terms and phrases used in this document, and variations thereof, unlessotherwise expressly stated, should be construed as open ended as opposedto limiting. As examples of the foregoing: the term “including” shouldbe read as meaning “including, without limitation” or the like; the term“example” is used to provide example instances of the item indiscussion, not an exhaustive or limiting list thereof; the terms “a” or“an” should be read as meaning “at least one,” “one or more” or thelike; and adjectives such as “conventional,” “traditional,” “normal,”“standard,” “known” and terms of similar meaning should not be construedas limiting the item described to a given time period or to an itemavailable as of a given time, but instead should be read to encompassconventional, traditional, normal, or standard technologies that may beavailable or known now or at anytime in the future. The term comprisingis intended herein to be open-ended, including not only the recitedelements, but any additional elements as well. Likewise, where thisdocument refers to technologies that would be apparent or known to oneof ordinary skill in the art, such technologies encompass those apparentor known to the skilled artisan now or at any time in the future. To theextent applicable, the terms “first,” “second,” “third,” etc. herein aremerely employed to show the respective objects described by these termsas separate entities and are not meant to connote a sense ofchronological order, unless stated explicitly otherwise herein.

The term “coupled” refers to direct or indirect joining, connecting,fastening, contacting or linking, and may refer to various forms ofcoupling such as physical, optical, electrical, fluidic, mechanical,chemical, magnetic, electromagnetic, communicative or other coupling, ora combination of the foregoing. Where one form of coupling is specified,this does not imply that other forms of coupling are excluded. Forexample, one component physically coupled to another component mayreference physical attachment of or contact between the two components(directly or indirectly), but does not exclude other forms of couplingbetween the components such as, for example, a communications link(e.g., an RF or optical link) also communicatively coupling the twocomponents. Likewise, the various terms themselves are not intended tobe mutually exclusive. For example, a fluidic coupling, magneticcoupling or a mechanical coupling, among others, may be a form ofphysical coupling.

The terms “substantially” and “about” used throughout this disclosure,including the claims, are used to describe and account for smallfluctuations, such as due to variations in processing. For example, theycan refer to less than or equal to ±5%, such as less than or equal to±2%, such as less than or equal to ±1%, such as less than or equal to±0.5%, such as less than or equal to ±0.2%, such as less than or equalto ±0.1%, such as less than or equal to ±0.05%.

The presence of broadening words and phrases such as “one or more,” “atleast,” “but not limited to” or other like phrases in some instancesshall not be read to mean that the narrower case is intended or requiredin instances where such broadening phrases may be absent. The use of theterm “component” does not imply that the elements or functionalitydescribed or claimed as part of the component are all configured in acommon package. Indeed, any or all of the various elements of acomponent, including structural elements, can be combined in a singlepackage or separately maintained and can further be distributed inmultiple groupings or packages.

Additionally, the various examples set forth herein are described interms of example diagrams and other illustrations. As will becomeapparent to one of ordinary skill in the art after reading thisdocument, the illustrated examples and their various alternatives can beimplemented without confinement to the illustrated examples. Forexample, block diagrams and their accompanying description should not beconstrued as mandating a particular architecture or configuration.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

We claim:
 1. A method comprising: attaching a single DNA templatemolecule to each of a plurality of attachment elements on a samplecontainer, wherein the average distance between adjacent elements isless than Abbe's limit; applying a stochastic photo-switching chemistryto all of the molecules at the same time to cause the attached moleculesto fluoresce in on and off events in up to four different colors bystochastic photo-switching; and imaging the on and off events in a colorchannel for each color in real-time as the on and off events areoccurring for the attached molecules.
 2. The method of claim 1, whereineach of the plurality of attachment elements on the sample container iswithin a field of view of an imager used to image the on and off eventssuch that imaging of the on and off events occurs at the same time forthe attached molecules at the plurality of attachment elements.
 3. Themethod of claim 1, applying a stochastic photo-switching chemistry toall of the attached molecules at the same time comprises applying astochastic optical reconstruction microscopy, a DNA Points Accumulationfor Imaging in Nanoscale Topography, or a direct stochastic opticalreconstruction microscopy stochastic photoswitching chemistry to all ofthe molecules at the same time.
 4. The method of claim 1, wherein due tothe stochastic nature of the photo switching, the probability that an onevent for a given base for a given molecule will occur at the same timeas an on event for the same base at a molecule adjacent to the givenmolecule is less than about 0.5%.
 5. The method of claim 1, whereinconcentrations of reagents for the stochastic photo switching aresufficient such that the probability that an on event for a given basefor a given molecule will occur at the same time as an on event for thesame base at a molecule adjacent to the given molecule is less thanabout 0.5%.
 6. The method of claim 1, further comprising, controlling arate at which the on and off events occur to control a probability thatan on event for a given base for a given molecule will occur at the sametime as an on event for the same base at a molecule adjacent to thegiven molecule.
 7. The method of claim 6, wherein controlling the rateat which the on and off events occur comprises adjusting concentrationsof nucleotides and enzyme in the stochastic photo-switching chemistry.8. The method of claim 6, wherein controlling the rate at which the onand off events occur comprises adjusting the on and off times so thatthe probability that an on event for a given base for a given moleculewill occur at the same time as an on event for the same base at amolecule adjacent to the given molecule is lower than a determined errorrate in a sequencing application in which the method is applied.
 9. Themethod of claim 1, further comprising, determining whether anillumination intensity of a detected on event in a color channel isgreater than a predetermined threshold.
 10. The method of claim 1,further comprising, determining whether a spot size of a detected onevent in a color channel is greater than a predetermined threshold. 11.The method of claim 1, wherein the average distance between adjacentelements is less than about 20 nm.
 12. The method of claim 1, whereinthe average distance between adjacent elements is within a range ofabout 2 nm to about 20 nm.
 13. An imaging system comprising: a samplecontainer comprising a plurality of attachment elements wherein a singleDNA template molecule is attached to each of the attachment elements,and further wherein the average distance between adjacent attachmentelements is less than Abbe's limit; and an imager positioned to imagephoto-switching occurring at the plurality of attachment elements bycapturing on and off events in a plurality of color channels at the sametime as the on and off events are occurring for the attached moleculeswhen a stochastic photo-switching chemistry is applied to all of theattached molecules at the same time causing the attached molecules tofluoresce in the on and off events in up to four different colors. 14.The imaging system of claim 13, wherein the sample container comprises aflowcell that comprises the plurality of attachment elements at aplurality of sample locations.
 15. The imaging system of claim 13,wherein each of the plurality of attachment elements on the samplecontainer is within a field of view of the imager used to image thephoto-switching such that the capturing of the on and off events occursat the same time for the attached molecules at the plurality ofattachment elements.
 16. The imaging system of claim 13, the stochasticphoto-switching chemistry applied to all of the attached molecules atthe same time comprises a stochastic optical reconstruction microscopy,a DNA Points Accumulation for Imaging in Nanoscale Topography, or adirect stochastic optical reconstruction microscopy stochasticphotoswitching chemistry.
 17. The imaging system of claim 13, whereinconcentrations of reagents for the stochastic photo switching aresufficient such that the probability that an on event for a given basefor a given molecule will occur at the same time as an on event for thesame base at a molecule adjacent to the given molecule is less than0.5%.
 18. The imaging system of claim 13, wherein a rate at which the onand off events occur yields a probability that an on event for a givenbase for a given molecule will occur at the same time as an on event forthe same base at a molecule adjacent to the given molecule is lower thana determined error rate in a sequencing application in which the methodis applied.
 19. The imaging system of claim 13, further comprising,determining whether an illumination intensity or a spot size of adetected on event in a color channel is greater than a predeterminedthreshold.
 20. The imaging system of claim 13, wherein the averagedistance between adjacent elements is less than about 20 nm.